IFN-γ stimulated murine and human neurons mount anti-parasitic defenses against the intracellular parasite Toxoplasma gondii

Dogma holds that Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites, even with IFN-γ stimulation. As several recent studies questioned this idea, here we use primary murine neuronal cultures from wild type and transgenic mice in combination with IFN-γ stimulation and parental and transgenic parasites to reassess IFN-γ dependent neuronal clearance of intracellular parasites. We find that neurons respond to IFN-γ and that a subset of neurons clear intracellular parasites via immunity regulated GTPases. Whole neuron reconstructions from mice infected with parasites that trigger neuron GFP expression only after full invasion reveal that ~50% of these T. gondii-invaded neurons no longer harbor parasites. Finally, IFN-γ stimulated human pluripotent stem cell derived neurons show an ~50% decrease in parasite infection rate when compared to unstimulated cultures. This work highlights the capability of human and murine neurons to mount cytokine-dependent anti-T. gondii defense mechanisms in vitro and in vivo.

In the current study Chandrasekaran et al. stimulated primary murine neurons with IFN-γ to characterize neuronal immune responses. They isolated neurons from wild-type and transgenic mice and infected them in vitro with parental and transgenic parasites to reconsider IFN-γ dependent neuronal parasite clearance. With the similar mechanism as macrophages, IFN-γ stimulated neurons cleared the parasites via immunity regulated GTPases. Next, neurons were isolated from infected mice, resulting in neuronal GFP expression after full invasion, suggested that about 40% of the T. gondiiinvaded neurons eliminated the parasites. Also, human stem cell derived neurons reduced parasite infection rate by about 50% upon IFN-γ stimulation. This study describes the first time that neurons are able to eliminate T. gondii in an IFN-γ dependent manner.The results are very interesting and significant for the Toxoplasma field. The study is well designed, methodology is sound, the data analysis and interpretation is correct. In the present study, Koshy and colleagues have analyzed immune responses in neurons against an important human and animal intracellular pathogen, Toxoplasma. They developed a new transgenic parasite line expressing Cre fused with a dense granule protein, allowing them to identify Toxoplasmainfected/post-infected in the Cre reporter cells or mice. Using the Cre-expressing Toxoplasma, they found that neurons in mice expressed GFP without the parasites, suggesting that parasites have infected but been cleared. They further analyzed the mechanisms of parasite clearance and found that interferon-γ-inducible GTPases such as IRGs and GBPs are important for IFN-γ-mediated parasite clearance from neurons. Furthermore, the authors also found that IFN-γ stimulated human neurons clear the parasite.
(Major point) The authors described "Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites,,," as dogma. However, given that IFN-γ-dependent inhibition of the parasite proliferation in human neurons has been already reported (PMID: 31119110), the IFN-γ-induced anti-Toxoplasma immune response in neurons described in the present study is conceptually no longer novel.

(Minor point)
1)The authors should describe the strategy to generate Gra16-Cre Toxoplasma in Methods. The Cre possessed NLS signal? How the Cre fused with Gra16? What is the drug resistance marker for selection? 2) Fig. 5; the authors discriminate infected or uninfected neurons only by GFP-negative "hole" formation with Hoechst. The authors should have tested by indirect immuno-staining parasite proteins for the presence in the "hole". Reviewer #3 (Remarks to the Author):

Major findings
The study demonstrates that primary murine neurons can effectively control the intracellular parasite T. gondii after stimulation with IFNg. This finding clarifies previous conflicting results in the literature about the role of IFNg in controlling T gondii infection within neurons. It also extends previous studies demonstrating that neurons stimulated with interferons can control viral infections to now include protozoan parasites.
The study is largely confirmatory of mechanisms that have been demonstrated in other murine cells. The authors provide some evidence that clearance in IFNg stimulated neurons is due to expression and recruitment of IRGs. This is not surprising as the IRG system constitutes the major mechanism of control in T gondii in IFNg stimulated cells of multiple lineages in rodents.

Major points
1) The reduction in neuronal infection after IFNg stimulation is modest -showing only 25% reduction at 24 hrs. This result contrasts with other cell types including astrocytes where clearance rates are much more dramatic. So although the findings suggest neurons can exert some control over infection after IFNg stimulation, it is not clear how important the role of neuron control of infection is in vivo -despite some evidence that it can occur ( Fig 5).
2) The authors also show modest control of intracellular T gondii in human neurons treated with IFNg, although there is no insight into the mechanism of clearance. This greatly diminishes the significance of the findings that remain largely observational.
3) The dense granule protein being used as a fusion should be referred to by name in the text -rather than being anonymous. The export of this protein is somewhat surprising as fusion of globular proteins to GRAs normally completely blocks export (and ablates function). The authors should provide some estimate of how effectively this protein gets exported-independent of the Cre mediated activation of GFP. The large percentage of GFP+ cells that are not infected suggest the background is rather high in this assay. Although the authors suggest that this is due to division of fibroblasts after infection, the evidence for this is indirect. If the authors are convinced that the II-GCre line only marks infected neurons, they should present these data and omit the confusing results that occur in dividing cells.
4) The use of type III parasites expressing ROP18 and Irgm1/3 KO neurons provides partial evidence for the role of the IRG system in the control of T gondii infection in neurons. However, these experiments are lacking an important control. Do IFNg treated neurons clear type III parasites that do not express ROP18? Such data should be included in

Reviewer #1
It was previously shown that "immunologically incompetent" neurons are the strategic location in the CNS where the intracellular parasite Toxoplasma gondii persist. Early studies applied IFN-γ stimulation to prove that neurons are not able to clear the parasites directly.
In the current study Chandrasekaran et al. stimulated primary murine neurons with IFN-γ to characterize neuronal immune responses. They isolated neurons from wild-type and transgenic mice and infected them in vitro with parental and transgenic parasites to reconsider IFN-γ dependent neuronal parasite clearance. With the similar mechanism as macrophages, IFN-γ stimulated neurons cleared the parasites via immunity regulated GTPases. Next, neurons were isolated from infected mice, resulting in neuronal GFP expression after full invasion, suggested that about 40% of the T. gondii-invaded neurons eliminated the parasites. Also, human stem cell derived neurons reduced parasite infection rate by about 50% upon IFNγ stimulation. This study describes the first time that neurons are able to eliminate T. gondii in an IFN-γ dependent manner. The results are very interesting and significant for the Toxoplasma field. The study is well designed, methodology is sound, the data analysis and interpretation is correct.
We appreciate Reviewer #1s recognition that we are significantly changing the current Toxoplasmaneuron paradigm.
Minor: Fig.1C the x axis is difficult to read Thank you for pointing this out, we have made the font bigger and bolded it.

Figures legends contain some typos
We apologize for not catching the typos and believe we have now fixed all of them.

Reviewer 2
In the present study, Koshy and colleagues have analyzed immune responses in neurons against an important human and animal intracellular pathogen, Toxoplasma. They developed a new transgenic parasite line expressing Cre fused with a dense granule protein, allowing them to identify Toxoplasmainfected/post-infected in the Cre reporter cells or mice. Using the Cre-expressing Toxoplasma, they found that neurons in mice expressed GFP without the parasites, suggesting that parasites have infected but been cleared. They further analyzed the mechanisms of parasite clearance and found that interferon-γ-inducible GTPases such as IRGs and GBPs are important for IFN-γ-mediated parasite clearance from neurons. Furthermore, the authors also found that IFN-γ stimulated human neurons clear the parasite. We appreciate this summary.
1. The authors described "Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites,,," as dogma. However, given that IFN-γ-dependent inhibition of the parasite proliferation in human neurons has been already reported (PMID: 31119110), the IFN-γ-induced anti-Toxoplasma immune response in neurons described in the present study is conceptually no longer novel.
Reviewer 2's major concern is that the novelty of this submission is compromised because "IFN-γinduced anti-Toxoplasma immune response in [human] neurons" has already been described (PMID: 31119110). In this context, it is important to recognize two things about Bando et al Front Cell Infect Micro 2019: 1. The paper uses a variety of cell lines to model the role of IFN-γ +/-IL-1B in controlling T. gondii. One of these cell lines is a neuroblastoma cell line, which the paper cites as a CNS cell line. Neuroblastomas are embryonal tumor cells of the autonomic nervous system and the majority arise in the adrenal glands that sit above the kidney. It's a simple mistake-but these are not equivalent to cortical neurons. 2. The authors did use primary human neurons but these cells were co-cultured with infected monocytesi.e., the neurons were not directly infected. In fact, it is unclear if the neurons ever became infected. In short, the authors do not assess the impact of their stimuli on the ability of neurons to control growth of T. gondii. Thus, to the best of our knowledge, our work is the only study that directly and unequivocally addresses the effect of IFN-γ on human neuron defenses against T. gondii. We did not cite this paper previously, but we have now cited the paper (lines 318-324) and outlined the differences between the prior work and the current submission.  Fig 2A).
Here we provide experimental evidence that explains how those authors reached their conclusion based on the parasite strain used and highlight that the more common, less virulent strains are susceptible to cell-intrinsic, IFN-γ-dependent neuronal defense mechanisms. Thus, our work resolves these differences. (Fig 5) establish that a mechanism of control that is independent of cytotoxicity of infected neurons exists and that IFN-γdependent, cell-intrinsic clearance is more likely. In addition, unlike the Salvioni paper, the Cre reporter mice we use only express the class I MHC H-2 L d . Our data, therefore, are the first to suggest that productive CD8 + T cell-neuron interactions also occur in wild type B6 mice. We have added these important points to the discussion.

A recent paper by the Blanchard group indicates that MHC class I on neurons helps with parasite control (Salvioni et al Cell Reports 2019) but it is unclear whether that control is secondary to cytolysis of infected neurons; induction of IFN-γ; or even non-cytolytic perforin-dependent clearance by CD8 T cells (Suzuki et al 2010). Our data showing clearance infected neurons in vivo
(Minor point) 1)The authors should describe the strategy to generate Gra16-Cre Toxoplasma in Methods. The Cre possessed NLS signal? How the Cre fused with Gra16? What is the drug resistance marker for selection? We apologize for the limited way in which we described the generation of the Gra16-Cre strain. We have now provided more details about generating these parasites, including referencing the original ptoxofilin-Cre plasmid that was modified to create a pgra16-Cre plasmid and identifying the selection marker (hpt).
2) Fig. 5; the authors discriminate infected or uninfected neurons only by GFP-negative "hole" formation with Hoechst. The authors should have tested by indirect immuno-staining parasite proteins for the presence in the "hole". We appreciate the Reviewer's concern. Staining 200 um thick brain sections with traditional antibodies is extremely challenging, especially when matched with the need to preserve intrinsic GFP signal. To address the Reviewer's concern, we have stained thick sections with both Hoechst and anti-T. gondii antibodies to show that what we identify as parasites by Hoechst directly matches with anti-T. gondii staining (updated Fig 5 and Fig S4A, B). In addition, we have provided more examples of the correlation between Hoechst and "the hole" (Fig S4C).
3) Fig. 3; what is IRG KO? Irgm1/m3 KO? 4) Fig. S3; what is Pru::TCre? Is this R-Cre? Thank you for catching these inconsistencies. Through various drafts we had used IRG KO and Pru::TCre to denote Irgm1/m3 KO and RCre, respectively. We thought we had caught all of these inconsistencies but clearly we missed several. We have fixed these inconsistencies. Fig. S4; the generation of mice is not worthwhile making a figure.

5)
We have removed the figure.

Reviewer 3
The study demonstrates that primary murine neurons can effectively control the intracellular parasite T. gondii after stimulation with IFNg. This finding clarifies previous conflicting results in the literature about the role of IFNg in controlling T gondii infection within neurons. It also extends previous studies demonstrating that neurons stimulated with interferons can control viral infections to now include protozoan parasites. We appreciate that Reviewer 3 acknowledges that the body of work in this paper has rigorously addressed how IFN-γ affects control of T. gondii within neurons, thereby resolving a long-standing issue in the field, which also has broader relevance to other neurotropic infections.
The study is largely confirmatory of mechanisms that have been demonstrated in other murine cells. The authors provide some evidence that clearance in IFNg stimulated neurons is due to expression and recruitment of IRGs. This is not surprising as the IRG system constitutes the major mechanism of control in T gondii in IFNg stimulated cells of multiple lineages in rodents.

It is important to recognize that, like anti-viral responses, there appears to be cell-specific variability in the anti-parasitic responses in both human (Fisch et al 2019) and murine cells. For example, while in
vivo work showed that Irgd/IRG-47 KO mice are more susceptible to T. gondii (Taylor et al 2001),

Butcher et al 2005 showed that IFN-γ-dependent control of T. gondii in murine macrophage does not require Irgd/IRG-47. Given the work showing that neurons have unique responses to IFN-γ (Rose et al 2007) coupled with an understanding of cell-specific IFN-γ responses, there is no reason to expect neurons to clear intracellular parasites by the mechanism used in non-neuronal cells. The potential for neurons to use alternative mechanisms for clearance is emphasized by the fact that autophagy is particularly highly utilized in neuronal clearance of some viruses (Orvedahl et al 2010). The options for
how IFN-γ might influence neuron control of T. gondii is broad [e.g., decreased attachment/invasion, killing of parasites via NO or tryptophan sequestration (nutritional immunity), and autophagy]. For these reasons, the data showing that IFN-γ leads to clearance of intracellular parasites (GCre parasites) by the IRGs are of particular importance. Basically, it's better to experimentally test and verify than to guess. This is the first time that IRG recruitment has been shown in neurons.

Major points
1) The reduction in neuronal infection after IFNg stimulation is modest -showing only 25% reduction at 24 hrs. This result contrasts with other cell types including astrocytes where clearance rates are much more dramatic. So although the findings suggest neurons can exert some control over infection after IFNg stimulation, it is not clear how important the role of neuron control of infection is in vivo -despite some evidence that it can occur (Fig 5). We agree with Reviewer 3 that the difference between in vitro IFN-γ mediated neuron clearance and other cells such as astrocytes and macrophages is notable. Where we disagree with Reviewer #3 is with the discounting of our in vivo data as lacking importance. As we note in the Discussion, the increased clearance rate of neurons in vivo (50% vs. 20-25%) suggests other factors may be at play, including other mechanisms of clearance, other cells making neuronal clearance efficient, etc. Thus, cell-specific knockouts of a single pathway in mice may not show major differences but that may be because other pathways are upregulated. Using much more expansive blockade (cell-specific IFN-γ receptor KOs) is likely to lead to a lack of parasite control but potentially for many reasons, not just neuron-specific clearance of intracellular pathways. In addition, we would like to highlight that if one wants to understand how to make "curative" therapies for T. gondii, then we need to understand why T. gondii persists in neurons. If we can understand why neurons are less effective than other cells at clearing T. gondii, then perhaps we can find ways to remove those neuron-specific blocks. Such a possibility would be akin to how understanding how PD1 and CTLA4 repression of T cell response had led to a revolution in cancer therapeutics. We have modified the discussion to emphasize these important implications.
For these reasons, like Reviewer #1, we strongly believe that having moved from a state of "IFN-γ stimulated neurons do not clear intracellular parasites" to clear evidence that IFN-γ stimulation leads to clearance of parasites in some neurons in vitro and in vivo is a substantial step to take for a single paper. [Redacted] 2) The authors also show modest control of intracellular T gondii in human neurons treated with IFNg, although there is no insight into the mechanism of clearance. This greatly diminishes the significance of the findings that remain largely observational. We respectfully disagree that showing that IFN-γ stimulated human neurons have anti-parasitic responses without identifying the mechanism diminishes the significance. Given the range of possible mechanisms, an in-depth study to define the mechanisms that may be at play (e.g., decreased attachment/invasion, killing of parasites via NO or tryptophan sequestration (nutritional immunity), and autophagy) is beyond the scope of this paper. To rigorously dissect out the mechanism(s) at play will be a paper unto itself.
In addition, many publications have shown that human non-immune cells show a rate of 5-30% for markers of clearance (Fisch et al 2019, Selleck et al 2015, Clough et al 2016.

Given this body of work, a 50% reduction in neuron infection rate is quite significant for a non-immune cell, especially as our study is the first to address the impact of IFN-γ on human neurons (see comment to Reviewer #2).
3) The dense granule protein being used as a fusion should be referred to by name in the text -rather than being anonymous. The export of this protein is somewhat surprising as fusion of globular proteins to GRAs normally completely blocks export (and ablates function). The authors should provide some estimate of how effectively this protein gets exported-independent of the Cre mediated activation of GFP. The large percentage of GFP+ cells that are not infected suggest the background is rather high in this assay. Although the authors suggest that this is due to division of fibroblasts after infection, the evidence for this is indirect. If the authors are convinced that the II-GCre line only marks infected neurons, they should present these data and omit the confusing results that occur in dividing cells. We apologize for this confusion. We have named GRA16 in the text. How efficiently a Gra16::fusion is exported depends on the fusion protein, which is detailed in Bracha et al bioRxiv 2018. We chose to use Gra16 as the fusion partner for Cre based upon these data (which were communicated to us personally by Lilach Sheiner). In addition, sensitive reporter systems often reveal biology not observable by more "standard" approaches. For example, until -lactamase was fused to toxofilin (Lodoen et al 2010), it was unclear if toxofilin was released into the host cell, though this possibility was suspected (Bradley et al 2005). Similarly, the concept of T. gondii injecting cells without invading them was not recognized until the advent of the T. gondii-Cre parasites (Koshy et al 2010;Koshy et al 2012).
The II-GCre line only marks infected neurons, which is highlighted by 98% +/-0.62% of the GFP + neurons harboring parasites when no there is no IFN-γ pre-stimulation. Conversely, for II-RCre parasites, which can inject Cre into cells without invasion, only 67%+/-1.8% of GFP + neurons harbor parasites. We removed the data with the fibroblasts (though we hated to remove primary data.) 4) The use of type III parasites expressing ROP18 and Irgm1/3 KO neurons provides partial evidence for the role of the IRG system in the control of T gondii infection in neurons. However, these experiments are lacking an important control. Do IFNg treated neurons clear type III parasites that do not express ROP18? Such data should be included in Fig 4. We have added these data.